Isopropyl Î²-D-1-thiogalactopyranoside, abbreviated IPTG, is a molecular biology reagent. This compound is used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon. Unlike allolactose, the sulfur (S) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from “eating up” or degrading the inductant; therefore the IPTG concentration remains constant. For induction, a sterile 1 M solution of IPTG is typically added by 1:1000 dilution into a logarithmically growing bacterial culture. Different final concentration of IPTG may be used.
IPTG binds to the lac repressor and releases the tetrameric repressorÂ from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon, such as the gene coding for beta-galactosidase, a hydrolase enzyme that catalyzes the hydrolysis of Î²-galactosides into monosaccharides. One advantage of IPTG for in vivo studies is that since it cannot be metabolized by E. coli its concentration remains constant and the rate of expression of lac p/o-controlled genes, is not a variable in the experiment. IPTG intake is independent on the action of lactose permease, since other transport pathways are also involved.
In cloning experiments, colonies that have been transformed with the recombinant plasmid rather than a non-recombinant need to be identified. X-gal is a substance that can be metabolised by beta-galactosidase to produce a blue product. Thus cells expressing beta-galactosidase grown in the presence of X-gal and IPTG (to induce the expression) will turn blue. Where a DNA fragment has been inserted into the LacZ (one of the genes for beta-galactosidase) there will be no action upon X-gal and the cells will not turn blue, thus identifying the cells that carry recombinant plasmid rather than non-recombinant plasmid.
Many regulatory elements of the lac operon are used in inducible recombinant protein systems; IPTG is an effective inducer in the concentration range of 100 Î¼M to 1.5 mM. Concentration used depends on the strength of induction required, as well as the genotype of cells or plasmid used – if lacIq, a mutant that over-produces the lac repressor, is present, then a higher concentration of IPTG may be necessary.
Sources: Hansen LH, Knudsen S, SÃ¸rensen SJ
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